Purpose. To investigate structural and functional correlations in glaucoma patients using optic nerve head hemoglobin (ONH Hb) measurements as determined by automated colorimetric analysis of conventional retinography. Methods. We prospectively enrolled healthy participants and glaucomatous patients with a wide range of disease stages. All participants underwent visual field (VF) testing (standard automated perimetry, SAP), color fundus imaging (mydriatic retinography), and peripapillary retinal nerve fiber layer (pRNFL) assessment through spectral-domain optical coherence tomography (SD-OCT). Software Laguna ONhE was used to estimate the amount of ONH Hb and to determine the glaucoma discriminant function (GDF) index. Scatter plots were constructed, and regression analysis was used to investigate the correlations between GDF, average pRNFL thickness, and VF mean deviation (VFMD) index values. A secondary analysis was performed to compare each parameter between three different glaucoma groups divided according to VFMD values (mild, >−6 dB; moderate, −6 to −12 dB; and advanced, <−12 dB). Results. One hundred ninety-six eyes from 123 participants (69 with glaucoma and 54 controls) were enrolled. Overall, all parameters evaluated differed significantly between glaucomatous and control eyes (). The comparison of each parameter according to groups of disease stages revealed significant differences between controls and each of the glaucomatous groups (). More pronounced changes in GDF values were observed in early disease stages. We found significant nonlinear correlations between GDF and VFMD values (R² = 0.295, ) and between pRNFL thickness and VFMD (R² = 0.598, ). A linear correlation was found between GDF and pRNFL thickness values (R² = 0.195, ). Conclusion. Our results showed significant associations between ONH Hb values and both structural and functional damage in glaucoma obtained by SD-OCT and SAP, respectively. The nonlinear correlation we found and the GDF behavior along different disease stages suggest that ONH Hb levels’ reduction may precede visual function changes in early glaucoma stages.
1. Introduction
Glaucoma consists in the main cause of irreversible blindness worldwide [1]. The disease is considered as a progressive and chronic optic neuropathy, characterized by specific changes on the optic nerve head (ONH), peripapillary retinal nerve fiber layer (pRNFL), and visual field (VF) [2, 3]. Disease control and blindness prevention are strictly related to early diagnosis [2, 3]. However, the diagnosis of glaucoma can be challenging in the early stages of the disease, especially for general ophthalmologists [4, 5]. Taking this into account, it is essential to perform the correlation between structural and functional changes. Anatomical evaluation can be performed through stereoscopic retinography [6] and automated quantitative exams such as optical coherence tomography (OCT) which were developed with the aim of contributing to the diagnosis of the disease [7–9]. Nevertheless, the high cost can represent a limitation to the access of the referred diagnostic tools.
Considering the pathological mechanisms of glaucoma, vascular dysfunction has been related to the optic nerve glaucomatous lesion [10]. The access to these vascular changes can be achieved through some diagnostic tests. At first, one can mention the evaluation of the ocular blood flow, through nearby vessels, using echo Doppler [11]. Other tests were developed to measure oxygen concentration in the optic nerve [12], blood flow, and vascular structure with the emergence and clinical application of OCT angiography (OCT-A) [13].
Previous studies have evaluated the hypothesis of a relationship between tissue perfusion and the level of hemoglobin (Hb) and oxygenation. Tissues with adequate perfusion demonstrate a good level of Hb, whereas low levels occur in tissue loss [14, 15]. Some studies have proposed a simple method for measuring hemoglobin levels in the ONH, assessing conventional retinography through automated colorimetric analysis, using software Laguna ONhE [16–19]. These preceding data have demonstrated that lower levels of optic nerve head hemoglobin (ONH Hb) are found in patients with established glaucoma, along with high reproducibility results, both in glaucomatous and nonglaucomatous eyes [15].
All these considered, we sought to investigate the correlation between the levels of ONH Hb, assessed by automated colorimetric analysis, and the levels of structural and functional damage, obtained by spectral-domain optical coherence tomography (SD-OCT) and standard automated perimetry (SAP), respectively, in glaucomatous patients.
2. Methods
This study protocol, according to the tenets of the Declaration of Helsinki, was approved by the ethics committee and the institutional review board of the Federal University of São Paulo (CEP: 4.055.180). Written informed consent was obtained by all participants prior to enrollment and examination.
2.1. Participants
In this observational cross-sectional study, we included consecutive healthy individuals and patients with primary open-angle glaucoma attending to the Glaucoma Sector of Hospital Medicina dos Olhos (São Paulo, Brazil) between May 2020 and January 2021.
Glaucoma was defined as the presence of glaucomatous optic neuropathy (GON) associated or not with the corresponding VF alteration. The criteria used to define the disease were the same as those used by our research group in previous studies [20, 21]. GON was considered in the presence of a vertical cup-to-disc ratio (VCDR) greater than or equal to 0.6, asymmetry of VCDR between the eyes (greater than 0.2), detection of localized or diffuse pRNFL defects, or neuroretinal rim defects, without other pathologies that could explain these changes. We adopted VF glaucomatous defect in the SAP (Humphrey SITA—Standard 24-2, Carl Zeiss Meditec, Dublin, CA), if there were, on the pattern deviation plot, three or more points in clusters with a probability of less than 5% (points directly above and below the blind spot or on the edge of the field were excluded), a pattern standard deviation index with a probability of less than 5%, or the result outside the normal limits on glaucoma hemifield test.
The following exclusion criteria were adopted: age ≤18 years, previous ocular trauma or posterior segment intraocular surgery, significant media opacity, difficulty in performing the exams, diagnosis of primary angle closure or secondary glaucoma, and presence of ocular diseases other than glaucoma that could influence the results, such as diabetic or hypertensive retinopathy and macular edema.
Regarding the control group, nonglaucomatous patients were included, demonstrating normal appearance of the optic disc, such as a VCDR less than 0.6, absence of defects on the neuroretinal rim or pRNFL, and intraocular pressure (IOP) less than 21 mmHg, without treatment [20].
2.2. Study Protocol
Complete ophthalmological examination was performed in all participants. This evaluation included clinical history, best-corrected visual acuity, slit-lamp biomicroscopy, IOP measurement with Goldmann applanation tonometry, gonioscopy, ultrasound pachymetry, dilated fundus examination, VF testing (Humphrey SITA—Standard 24-2, Carl Zeiss Meditec, Dublin, CA), color fundus imaging (mydriatic fundus retinography Canon CR-2; Canon, Tokyo, Japan), and pRNFL and topographic ONH measurements based on SD-OCT (RTVue-100 OCT; Optovue Inc., Fremont, CA).
Accepted reliability indices for this protocol include patients’ experience in performing VF testing (at least 3 previous exams). Patients were excluded from the study if the exams presented >15% false positives or >33% loss of fixation or false negative. Additionally, during SAP review, the exam was eliminated in the presence of some artifacts such as edge defects, inattention or loss of fixation, fatigue effect, or alterations indicative of pathologies other than glaucoma.
The color fundus retinography was then analyzed by Laguna ONhE software. The full description of the program was presented in a previous study [16]. Summarizing, Laguna ONhE analyzes conventional fundus photographs to measure the amount of ONH Hb. Software considers three spectral components of ONH photographs: red, green, and blue. The red component is reflected by ONH areas with a high Hb content. On the contrary, a smaller proportion of the red light, compared to green and blue components, is reflected in areas with low Hb content. The analysis of various formulas, based on the reflected amounts of red, green, and blue light, was almost linearly proportional to the amount of Hb present [16]. Figure 1 demonstrates examples of patients with normal and glaucomatous papilla and the respective pseudo-images indicating the Hb levels. Finally, software determines an index of glaucoma discriminant function (GDF), based on colorimetric analysis and OHN Hb levels [16]. This index was performed by dividing the ONH into 3 concentric rings, and each one of them was divided into 8 parts, a total of 24 sectors. The sectors that showed the greatest difference in the amount of Hb, between eyes of patients with glaucoma and control, were those located in the vertical region, especially sectors 8 and 20, as shown in a previous study. From this, the GDF takes into account the slope of the Hb amount with the mean of these specific sectors (8 and 20) presenting 89% sensitivity at 95% specificity [16].
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